We aim at identifying biomarkers that give us information about the physiological status of the female genital tract, in relation to HIV-1 and HPV infection.In addition, we focus on the intracellular molecular events that take place after infection of a cell with HIV-1, HPV or VSV.
The cervicovaginal fluid (CVF) is a body fluid originating from the vagina and ectocervix that has some marked benefits for the study of biomarkers of the female genital tract.
In this fluid, the biomarker is not diluted in a large volume (eg. plasma: dilution in several liters of blood) allowing for a higher sensitivity of detection. Moreover, the CVF is mostly (although not exclusively) composed of secretions from the female genital organs, allowing for a higher specificity of detection. The fluid can also easily be sampled (eventually by the individual herself) which is an advantage, especially in regions where gynecologists and/or practitioners are scarce.
A major obstacle in the determination of biomarkers from CVF (and nearly all other body fluids) is the high intra- and interindividual variability between the samples. We developed a 2D-LC/MS methodology that allows for the identification of proteins from complex samples such as CVF. Comparison with other large-scale studies shows that a set of proteins makes up the ‘core CVF proteome’ that is present in nearly every sample, irrespective of the physiological status of the individual or the proteomics method used.
In addition, we have demonstrated in two recent differential studies that potential biomarkers for cervix cancer and for HIV-1 resistance can be found in CVF:
- Differential proteomics experiments in our lab showed that biomarkers for cervix carcinoma very probably reside in the CVF, since individuals who are in the precancerous stage (have intraepithelial lesions) show a higher abundance of several marker proteins, previously described to be involved in cancer. These biomarkers may improve current screening strategies for cervix cancer but may also pave the way for the development of self-diagnosis tests for the disease.
- In a second collaborative project, the CVF proteome from women, resistant to HIV, was compared to the CVF proteome from HIV sensitive women. We demonstrated up- and downregulation of proteolysis-related proteins, in de CVF from HIV resistant women. Our conclusion is that at least in part, a precise balance of proteases and protease inhibitors in CVF is needed to acquire the HIV resistant state.
Zegels et al., 2009, 2010
INTRACELLULAR ANTIVIRAL DEFENCE MECHANISMS
We focused on a specific set of proteins, known to have an important contribution in the antiviral defence against viruses.
APOBEC3G, TRIM5alpha, tetherin and the proviral protein LEDGF/p75: anti-HIV-1 activity
- During recent years, the intrinsic antiviral properties of APOBEC3G, TRIM5alpha and tetherin became has been intensively studied. As the three proteins have shown to have anti-HIV-1 activity, it is important to know the expression levels of APOBEC3G and TRIM5alpha in several different types of immune cells. We therefore developed a FACS assay that allows for quantification of these intracellular proteins in eg. T-cell types and macrophages. A similar assay was developed for the proviral protein LEDGF/p75.
- Real-time PCR and FACS analysis were used to analyse mRNA and protein levels respectively in PBMC and several PBMC subsets. Significantly reduced LEDGF/p75 protein levels were seen in CD4+ lymphocytes of HIV-1-exposed seronegative subjects (ESNs, known to be resistant to HIV-1) relative to healthy controls, whereas no differences were seen in APOBEC3G, TRIM5alpha, or tetherin expression. Untreated HIV-1-infected patients generally expressed higher mRNA and protein levels than healthy controls. Increased tetherin levels correlated with markers of disease progression: directly with the viral load and T cell activation and inversely with the CD4 count.
Relevant publications: Mous et al., 2011, 2012
PML and PML nuclear bodies: networking and antiviral activity
- PML nuclear bodies are a hub for numerous proteins and are involved in a range of functions such as transcriptional regulation, apoptosis, cell cycle control and antiviral activity. Moreover, the complex is highly dynamic and proteins such as CBP and Sp100 show a dynamic exchange between nuclear bodies and nucleoplasm. The involvement of PML-NBs in a large variety of processes has led to an enormous research interest in this protein which culminated in an overwhelming number of publications. Therefore an integrative global approach is essential to grasp the true complexity of the PML-NBs and to allow us to generate hypotheses based upon the vast amount of information available in distributed sources.
We therefore developed a curated network model of PML-NBs based on information extracted from protein interaction databases (MINT, IntAct, BIOGRID, HPRD and NPD) and fragmented information obtained through extensive literature search. For the compilation of the network we used the open source network analysis software Cytoscape.
Since a more than average amount of PML partners have been described in literature or have been predicted as SUMOylated (38%), we postulated that PML-NBs are a nuclear SUMOylation hotspot. The PML-NBs can thus be seen as a nuclear relay station where protein fractions involved in a variety of nuclear processes make a SUMOylation regulated functional switch.
- PML-NBs are targeted by entire viral particles, viral proteins or viral nucleic acids. Their possible anti-viral properties motivated researchers to investigate the interaction between PML-NBs and viruses in depth. Based on extensive literature data mining, we created a comprehensive PML-NB/virus crosstalk Cytoscape network, which groups not only the most common relations but also less well described findings. The network is easy to navigate and provides a biologically relevant overview which can help finding interesting case studies.
- We have set up an immunocytochemical study to investigate if PML-mediated resistance against VSV is conferred by the recruitment of any of the five VSV proteins from the cytoplasm to the nucleus. Unexpectedly, we did not see co-localization of VSV proteins with PML-NBs and we observed only minor changes in the subcellular distribution of the VSV proteins in a VSV resistant PML overexpressing cell line. These observations suggest an indirect role of PML in VSV resistance.
Relevant publications: Van Damme et al., ,2010, 2011.